Evaluation of Cyprinid Herpesvirus 2 Latency and Reactivation in Carassius gibel

Abstract

Cyprinid herpesvirus 2 (CyHV-2, species Cyprinid herpesvirus 2) causes severe mortality in ornamental goldfish, crucian carp (Carassius auratus), and gibel carp (Carassius gibelio). It has been shown that the genomic DNA of CyHV-2 could be detected in subclinical fish, which implied that CyHV-2 could establish persistent infection. In this study, the latency of CyHV-2 was investigated in the survival fish after primary infection. CyHV-2 genomic DNA was detected in multiple tissues of acute infection samples; however, detection of CyHV-2 DNA was significantly reduced in fish recovered from the primary infection on day 300 postinfection. No active viral gene transcription, such as DNA polymerase and ORF99, was detected in recovered fish. Following temperature stress, an increase of CyHV-2 DNA copy numbers and gene transcription were observed in tissues examined, which suggests that CyHV-2 was reactivated under stress. In addition, a cell line (GCBLat1) derived from the brain tissue from CyHV-2-exposed fish harbored CyHV-2 genome but did not produce infectious virions under normal culture conditions. However, CyHV-2 replication and viral gene transcription occurred when GCBLat1 cells were treated with trichostatin A (TSA) or phorbol 12-myristate 13-acetate (TPA). It suggests CyHV-2 can remain latent in vitro and can reactivate under stress condition.

Keywords: Cyprinid herpesvirus 2; cell line; gibel carp; latency; reactivation.

Figure 1

Detection of CyHV-2 DNA and gene transcription from moribund fish or surviving fish after primary infection with CyHV-2: Quantification of the CyHV-2 DNA in tissues from moribund fish (A) or surviving fish (C) by qPCR. White bars and black bars represent two moribund fish A1 and A2 in panel A and two surviving fish in panel C, respectively. CyHV-2 DNA copies were calculated according to the standard curve based on the series of diluted plasmids containing the DNA polymerase gene and were expressed as copies per µg of total DNA (copies/µg DNA). Data in A and C are presented as the mean of triplicates ± SD of each tested tissue sample from two representative fish. RT-PCR analysis of total RNA harvested from moribund fish (B) or surviving fish (D) tissues after primary infection with CyHV-2 in the presence (RT+) or absence (RT-) of reverse transcriptase: The amplicons of DNA pol (362 bp) and ORF99 (513 bp) are indicated. Lanes 1 to 6 represent the tissue of head-kidney, gills, midbrain, medulla oblongata, heart, and spleen, respectively. The amplicons of β-actin gene are used as an internal control to ensure that comparable levels of input RNA were used in RT-PCR. One representative experiment of three is shown.

Figure 2

Detection of CyHV-2 DNA and gene transcription in the moribund fish from temperature stress: (A) Quantification of the CyHV-2 DNA from three moribund fish suffered temperature stress by qPCR. White (1), gray (2), and black (3) bars respectively represent the different moribund fish. CyHV-2 DNA copies were calculated similarly, as described in Figure 1. The DNA copies shown are the mean of triplicates ± SD. (B) RT-PCR analysis of total RNA harvested from moribund fish in the presence (RT+) or absence (RT-) of reverse transcriptase: The amplicons of DNA pol (362 bp) and ORF99 (513 bp) are indicated. Lanes 1 to 6 respectively represent the tissue of the head, kidney, gills, midbrain, medulla oblongata, heart, and spleen, which were sampled from the same fish. The amplicons of β-actin gene are used as an internal control to ensure that comparable levels of input RNA were used in RT-PCR. One representative fish of three is shown.

Figure 3

Characteristics of the GCBlat1 cell line: (A) Morphology of GCBlat1 cells at passage 80. Scale bars = 100 μm. (B) Growth curves of the GCBlat1 cell line at passage 30 at 28 °C within 4 days. Y-axis represents cell numbers of the mean ± standard deviation of three independent experiments. (C) Morphological characteristics of chromosomes arrested in metaphase at passage 36. (D) Frequency distribution of chromosomes: The x-axis represents the number of chromosomes; the y-axis represents the number of cells analyzed.

Figure 4

Examination of CyHV-2 DNA and gene transcription in the GCBlat1 cell line: (A) PCR analysis of the CyHV-2 DNA from GCBlat1 cells at indicated passages. Amplicons of DNA pol (362 bp) and ORF 99 (513 bp) are indicated. (B) qPCR analysis of the CyHV-2 DNA in GCBlat1 cells at indicated passages: CyHV-2 DNA copies were calculated similarly as described above. The data shown are the mean of triplicates ± SD. (C) RT-PCR analysis of total RNA harvested from GCBlat1 cells of passage 32: Lane 1, β-actin; lane 2, DNA pol (362 bp); lane 3, ORF99 (513 bp); M, DNA ladder. One representative experiment of three is shown.

Figure 5

Examination of CyHV-2 DNA and gene transcription in GCBlat1 cells upon chemicals treatment: Cell viabilities of GCBlat1 cells were measured by an MTT assay following treatments with trichostatin A (TSA) (A) or phorbol 12-myristate 13-acetate (TPA) (B) (80, 40, 20, 10, and 5 μM) or dimethyl sulfoxide (DMSO) for 48 h. (C) qPCR analysis of the CyHV-2 DNA from GCBlat1 cells with TSA, TPA, DMSO, or without treatment (nc). CyHV-2 DNA copies were calculated similarly as described above. Data presented as the mean ± SD (n = 3). (D) RT-PCR analysis of total RNA harvested from treated and untreated GCBlat1 cells: The amplicons of DNA pol (362 bp) and ORF99 (513 bp) are indicated. The amplicons of β-actin gene are used as an internal control to ensure that comparable levels of input RNA were used in RT-PCR. M: DNA ladder; NC: negative control for PCR; lane 1: mock-treated GCBlat1 cells; lane 2: DMSO-treated GCBlat1 cells; lane 3: TPA-treated GCBlat1 cells; lane 4: TSA-treated GCBlat1 cells. One representative experiment of three is shown here. * p < 0.05